Low temperatures decrease the energy of motion of molecules, leading to increased stability and rigidity. For example, as temperature decreases proteins become less flexible, membrane lipids become less fluid, and secondary structures of DNA and
RNA become more stable. As a general mechanism, cold-adapted microorganisms increase the disorder within macromolecules to maintain fluidity or flexibility, and hence function at low temperatures (Feller 2007). In P. arcticus 273-4, a variety of adaptations are believed to facilitate the motion of biomolecules and cellular structures at low temperatures, and include amino acid composition, specific chaperone proteins, membrane components, and cell-wall structure.
Low temperatures reduce the activity of enzymes through decreased flexibility of protein structure. Cold adaptation of enzymes is commonly achieved in psy-chrophiles by reducing weak stabilizing interactions (ion pairs, hydrogen bonds, hydrophobic and intersubunit interactions), increasing solvent interactions with apolar or interior residues, reducing proline and arginine content, and/or clustering of glycine residues (Feller et al. 1996; Russell 2000). Consistent with these themes in amino acid alteration, the genes of P. arcticus 273-4 contain fewer hydrophobic and acidic residues, fewer proline residues, and more lysine and fewer arginine residues when compared to their homologs in the Swiss-Prot Database (Ayala-del-Rio et al., personal communication). Having fewer acidic or proline residues was the most common modification observed in Psychrobacter genes. Overall, 56% of the genes of P. arcticus 273-4 can be classified as "cold-adapted" by at least one of these measures (less hydrophobic; fewer proline residues; less aliphatic; fewer acidic residues; or fewer arginine and more lysine residues) and, on average, each of these cold adapted genes contain three of the five types of adaptations described above.
Protein chaperones have been repeatedly identified as important components of low-temperature growth in mesophilic and psychrophilic bacteria (Phadtare and Inouye 2004). Peptide chaperones such as GroEL/ES and peptidyl-prolyl cis-trans isomerases (PPIase) are thought to be important for promoting correct protein folding at low temperature (Strocchi et al. 2006). Of the protein chaperones present in the genome of P. arcticus 273-4, only clpB (a protein disaggregating chaperone) was up-regulated at low temperature, suggesting that aggregation of denatured peptides at low temperature may be a hurdle at subzero temperatures. Other heat shock proteins and PPIases (except oxidative stress chaperones) were up-regulated only during growth at warm temperatures in P arcticus 273-4. The amino acid changes that result in cold-adapted genes (as observed from genomic analyses) may have left Psychrobacter sp. dependent on the function of heat shock proteins during growth at the relatively mild temperatures of 22°C and 17°C near the upper end of their growth temperature range.
Low temperatures also stabilize the secondary structures of nucleic acids, leading to the inhibition of the processes of transcription, translation, and DNA replication. Cold-adapted microorganisms alleviate stress on these processes via RNA chaperones and specialized helicases (Jiang et al. 1997; Chamot and Owttrim 2000; Phadtare et al. 2002). RNA chaperones, such as cold-shock proteins (csp), are thought to prevent secondary structure formation in RNA, thereby ensuring successful translation of transcripts in conjunction with other cold-shock proteins such as DEAD box helicases (Whyte and Inniss 1992; Goldenberg et al. 1997; Lim et al. 2000; Iost and Dreyfus 2006). Likewise, increased expression of specific ribosomal proteins may contribute to low-temperature function of the ribosome, with a tradeoff in increased thermolability of that translational apparatus (Bayles et al. 2000). In P. arcticus 273-4, several cold-shock genes associated with molecular motion were upregulated during growth at low temperatures, including csdA (a DEAD-box helicase) and rbfA (a ribosome binding factor); however, cspA was constitutively expressed at all temperatures. Constitutive expression of the major cold-shock protein transcript may be the result of exposure to continuous cold temperatures in the permafrost. Interestingly, up-regulation of cspA was observed in the proteome (Bakermans et al. 2007); thus, regulation of CspA in Psychrobacter may involve posttranscriptional control of protein synthesis or degradation.
The fluidity of cell membranes can be maintained at low temperatures by increasing unsaturated lipids, decreasing acyl chain length and branch-chained lip-ids, or altering polar head groups and by producing compatible solutes (Russell 1990). For example, psychrophilic bacteria commonly increase the proportion of C181 and/or C16 fatty acids at low temperatures (Russell 1990, 1997). To ensure that membrane fluidity is maintained at low temperatures, Psychrobacter species contain two separate mechanisms for creating unsaturated fatty acids in membrane lip-ids: de novo synthesis and fatty acid desaturases. Indeed, increased expression of membrane fatty acid desaturases was observed during growth of P. arcticus 273-4 at low temperature. A previously unreported response to low temperatures was also observed in genes responsible for the dynamic growth and elasticity of the cell wall (Yao et al. 1999). Lytic transglycosylases and D-alanyl-D-alanine carboxypepti-dases were up-regulated during growth of P. arcticus 273-4 at low temperature. Regulation of cell-wall elasticity could play a major role in growth rate control at low temperatures. Because elastic materials stiffen in the cold, losing their resilience and resistance to stretching, Psychrobacter sp. may actively regulate the elasticity of the peptidoglycan wall to maintain the turgor pressure required for growth in the frozen conditions of the permafrost.
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